膜通透性檢測

腸道細(xì)菌及其毒素的移位可能是自身免疫性疾病發(fā)病的重要因素。這種猜測在一段時間內(nèi)被認(rèn)為是正確的。但是由于沒有合適的研究工具，因此沒有得到廣泛研究。事實(shí)上，研究表明壓力、手術(shù)和輕微胃腸道疾病等顯著增加了黏膜通透性。因此，這種情況下顯著增加了細(xì)菌從腸黏膜進(jìn)入人體的機(jī)率。

Chondrex公司提供FITC標(biāo)記的右旋糖苷及D-木糖檢測試劑盒用于評估體內(nèi)和體外大分子對半透膜的通透性。

通透性評估試劑盒

通透性評估試劑-熒光標(biāo)記的右旋糖苷

一般來說，分子通過腸道進(jìn)入身體發(fā)生在細(xì)胞間或通過細(xì)胞進(jìn)入體內(nèi)。熒光標(biāo)記的右旋糖苷提供了簡單，可靠的方法用于評估體外或體內(nèi)細(xì)胞旁通透性。另一種方法是，通過D-木糖測定跨細(xì)胞通透性，也就是說通過D-木糖作為被動載體介導(dǎo)小腸吸收。

FITC-標(biāo)記的右旋糖苷

一些研究表明壓力、手術(shù)和輕微胃腸道疾病等顯著增加了黏膜通透性。熒光標(biāo)記的右旋糖苷提供了簡單，可靠的方法用于評估半透膜功能。關(guān)于FITC-標(biāo)記的右旋糖苷的分子量，（ 40kDa）,有研究表明在大腸桿菌中大多數(shù)蛋白分子量為25-120kDa,這表明具有生物學(xué)意義的蛋白質(zhì)在該范圍內(nèi)。因此Chondrex提供40kDa FITC-右旋糖苷作為相關(guān)大分子標(biāo)記物，用于體外或體內(nèi)半透膜通透性研究。

檢測流程：

1）飼喂前和實(shí)驗(yàn)過程中，禁食4小時。

2）口服40 kDa FITC-Dextran (10 mg/250 µl/mouse)。

3）3小時后，收集血漿。

4）1:2 (或更大)用PBS稀釋血漿

5）取50-100ul上去到96孔板。讀取熒光值：Ex: 490 nm  Em: 530 nm

     用PBS稀釋的FITC- 右旋糖苷繪制的標(biāo)準(zhǔn)曲線

參考文獻(xiàn)：

1.Aoki S, Y.K., Yokoyama T, Nonogaki T, Iwasaki S, Mitsui T, Niwa S., Role of enteric bacteria in the pathogenesis of rheumatoid arthritis: evidence for antibodies to enterobacterial common antigens in rheumatoid sera and synovial fluids. Ann Rheum Dis., 55: 363-9 (1996).

2.Peltonen R, Kjeldsen-Kragh J, Haugen M, Tuominen J, Toivanen P, Førre O, Eerola E. Changes  of faecal flora in rheumatoid arthritis during fasting and one-year vegetarian diet. Br J Rheumatol, 33: 638-43 (1994).

3.Peltonen R, N.M., Helve T, Hänninen O, Toivanen P, Eerola E. Faecal microbial flora and disease activity in rheumatoid arthritis during a vegan diet. Br J Rheumatol, 36: 64-8 (1997).

4.Terato K, Harper DS, Griffiths MM, Hasty DL, Ye XJ, Cremer MA, Seyer JM. Collagen-induced arthritis in mice: synergistic effect of E.coli lipopolysaccharide bypasses epitope specificity in the induction of arthritis with monoclonal antibodies to type II collagen. Autoimmunity, 22:137-47 (1995).

5.Vaahtovuo J, Munukka E, Korkeamäki M, Luukkainen R, Toivanen P. Fecal microbiota in early rheumatoid arthritis. J Rheumatol, 35(8):1500-5 (2008).

6.Van der Heijden, IM Wilbrink B, Tchetverikov I, Schrijver IA, Schouls LM, Hazenberg MP, Breedveld FC, Tak PP. Presence of bacterial DNA and bacterial peptidoglycans in joints of patients with rheumatoid arthritis and other arthritides. Arthritis Rheum, 43:593-8 (2000).

7.Anderlik P, Szeri I, Bános Z, Barna Z. Bacterial translocation after cold stress in young and old mice. Acta Microbiol Hung, 37:289-94 (1990).

8.Velin AK, Ericson AC, Braaf Y, Wallon C, Söderholm JD. Increased antigen and bacterial uptake in follicle associated epithelium induced by chronic psychological stress in rats. Gut, 53:494-500 (2004).

9.Khalif IL, Quigley EM, Konovitch EA, Maximova ID. Alterations in the colonic flora and intestinal permeability and evidence of immune activation in chronic constipation. Dig Liver Dis, 37: 838-49 (2005).

10.Chong BE, Wall DB, Lubman DM, Flynn SJ. Rapid Profiling of E. coli Proteins Up to 500 kDa from Whole Cell Lysates Using Matrix-assisted Laser Desorption/Ionization time-of-flight mass Spectrometry. Rapid Commun Mass Spectrom, 11(17):1900-8 (1997).